Imaging of the medial rectus muscle predicts the development of optic neuropathy in thyroid eye disease

Goal of the study was to evaluate bony orbit remodeling and extraocular muscle (EOM) volume in thyroid eye disease (TED) and their role as predicting factors for development of dysthyroid optic neuropathy (DON). Orbital computed tomography of 92 patients with TED with (76 orbits) or without DON (98 orbits) were retrospectively evaluated. Orbits (n = 40) of subjects without TED served as controls. Measurements of the bony orbit as well as EOM volume were incorporated into a generalized linear mixed model to predict DON. The angle of the medial orbital wall was significantly smaller (p < 0.001) in patients with TED (− 2.3 ± 3.6°) compared to patients with TED + DON (1.0 ± 4.1°). Both groups differed significantly from controls (− 4.2 ± 2.7°). Bowing of the medial orbital wall correlated positively with muscle volume (r = 0.564; p < 0.001). Total EOM volume was significantly larger in TED + DON (7.6 ± 2.5cm3) compared to TED only (5.6 ± 3.0cm3; p < 0.001) or controls (2.6 ± 0.5cm3). Multivariate analysis revealed the medial rectus muscle volume (TED: 1.06 ± 0.48cm3 vs. TED + DON: 2.16 ± 0.84cm3) as the strongest predictor, achieving a specifity of 86.7% and a sensitivity of 73.7% in diagnosing DON in univariate analysis. Though characterized by a wide range of variability, increased medial rectus muscle volume is the strongest predictor for DON in our patient cohort with TED when analyzing a single muscle.

Of these 92 patients with clinically diagnosed TED, 49 patients (98 orbits, 26 male 72 female, mean age 49 ± 13.8 years) were allocated to the TED only group (without DON). DON was diagnosed in 43/92 patients (86 orbits). Of these, 76 orbits (59 female, 17 male, mean age 60 ± 12.5 years) were allocated to the TED + DON group. Optic nerve dysfunction was considered present if at least two of the following tests were impaired: visual acuity, pupillary light response, color vision/saturation and perimetry; patients with other causes for the impairment of visual function were not included in this population 10 . Patients with other causes for the impairment of visual function were not included in this population 10 . Visual acuity in logMAR was 0.51 ± 0.51 (0.00-3.00) in patients with DON, with two patients being able to only count fingers or to see hand movement. 30 eyes of 76 patients with DON (39.5%) had a relative afferent pupillary defect. Mean defect was 10.86 ± 7.22 dB using automated static perimetry.
None of the assessed patients had any prior ophthalmic or sinusal surgery. We furthermore analyzed the orbits of 20 subjects (40 orbits, 28 female, 12 male, mean age 57 ± 8.9 years), who served as control. These CT scans were performed to e.g. rule out intracranial hemorrhage or ischemia. None of these subjects had skull or face injuries, a history of thyroid or orbital disease or any other condition that could interfere with proper measuring.
The study protocol was approved and consent was waived by the local ethics committee (Landesärztekammer Rhineland-Palatine, #2021-15977). The study was conducted according to state laws. All research was performed in accordance with relevant local guidelines and/or regulations and in accordance with the declaration of Helsinki.
Radiologic analyses. All orbital CT scans were part of a routine clinical protocol performed on a Toshiba Aquilion 32 slice CT (Toshiba Medical Systems Group Co, Zoetermeer, The Netherlands). The acquisition parameters used were as follows: 120 kV, 200 mA, 210 mm display FOV, pitch of 21 and a slice thickness of 0.5 mm.
To ensure consistency throughout all measurements, the measurements were taken on reconstructed 2 mm slices to be either orthogonal to the optic nerve (muscle volume measurements) (Fig. 2) or parallel to the infraorbitomeatal line (bony orbit measurements) (Fig. 3).  www.nature.com/scientificreports/ Angular measurements. Orbital angles and length of the lateral orbital wall, as well as medial wall measurements were performed as described in recent studies 13,14 .
The apex point was defined as the anterolateral border of the groove in the sphenoid body formed by the intracavernous portion of the internal carotid artery (red cross in Fig. 3A). From this point, the orbital rim angle was measured on the height of the medial palpebral ligament (angle between green lines in Fig. 3B). The medial and lateral orbital wall angles were assessed on the image where the medial and lateral rectus muscle had the widest diameter as angular change between the orbital rim angle and the widest point of the orbital wall (Fig. 3C,D). The length of the lateral orbital wall was measured just below the anterior clinoid process (Fig. 3E).
To assess the anterior medial wall angle, a perpendicular line was drawn from the beginning of the medial orbit wall on the nasal bone (Fig. 3F, point 1) perpendicular to the interzygomatic line. This was considered line  www.nature.com/scientificreports/ A (Fig. 3F, A). Line B (Fig. 3F, B) was drawn, connecting point 1 with the point of maximum excursion of the medial wall (Fig. 3F, point 2). The resulting angle formed between line A and line B was considered the anterior medial wall angle (Fig. 3F, AMW). This angle was considered positive if the point 2 was lateral to line A or negative, if medial to it. Exophthalmos/Protrusion was measured as the distance between the interzygomatic line and posterior sclera just lateral of the optic nerve head (blue lines, Fig. 3F).
Volumetric analyses of the extraocular muscles. Volume of the extraocular muscles was measured using the closed polygon tool of OsiriX version 9.0 (Aycan Digitalsysteme GmbH, Würzburg, Germany) by outlining the profile of the muscles on coronal images. Due to difficulties to accurately discern the levator palpebrae from the superior rectus muscle, these muscles were evaluated as the superior muscle group.
Statistical tests. Multiple statistics were conducted with IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, N.Y., USA) using linear mixed models to compare muscle volumes and angles between patient groups and a generalized linear mixed model to predict DON from these measurements. All available variables that were distinctly different (p < 0.05) on univariate analysis were then encompassed in a multivariate model. No adjustment for multiple testing was done.

Results
Disease duration was significantly shorter and clinical activity score was significantly higher in patients with TED + DON compared to patients with TED only (disease duration: 2.1 ± 3.1 vs. 4.6 ± 5.2 years, p = 0.006; CAS: 1.9 ± 1.1 vs. 3.9 ± 1.2, p < 0.001). Likewise, ocular motility was restricted significantly stronger in patients with TED-DON compared to TED only (Table 1). Compared to patients with TED only, the medial, inferior, lateral, and the superior muscle group volume were significantly higher in the TED + DON group using the univariate mixed model analysis (all p < 0.001). Muscle volume of the TED group was significantly larger (p < 0.005) compared to the control group in all measured muscles, except for the lateral rectus muscle (p = 0.055, Table 1 The lateral wall angle, orbital rim angle, as well as the length of the lateral orbit wall did not differ markedly between the TED + DON, TED or TED + DON (ua) groups. Neither was there a difference when comparing the control group with the TED + DON group and the TED group.
Both types of angle measurement of the medial orbital wall showed the correlation between increased muscle volume and bowing of the medial wall. The medial rectus muscle had a slightly higher correlation (r = 0.564) compared to the other muscles or to the total muscle volume ( Table 2).
In the generalized linear mixed model, which contained all muscle measurements with distinct differences (i.e. p < 0.05) between the TED + DON and TED groups, the medial rectus muscle was the most influential variable regarding diagnostic accuracy (specificity: 86.7%, sensitivity: 73.7%). Adding all other muscles resulted in a specificity of 87.8% and a sensitivity of 77.6%. Further addition of the medial wall angle did not confer any diagnostic benefit.
A few subjects (n = 5, Fig. 5, marked red) stood out in that they had a higher MRV than that of the TED + DON group without, however, being affected by DON. There are also patients with DON (n = 6, Fig. 5, marked yellow) having a similar MRV and a smaller medial wall angle compared to patients with TED only.

Discussion
Radiological characteristics in patients with DON have been a major subject of research. Changes such as enlargement of the superior ophthalmic vein, anterior displacement of the lacrimal gland or the presence of an intracranial fat prolapse have been investigated [17][18][19] . The most thoroughly investigated radiological parameter aiding the diagnosis of DON is the increase of extraocular muscle size, ultimately leading to apical crowding and DON.
In the underlying study we have been able to confirm an increase of muscle volume especially that of the medial rectus muscle, to be the strongest predictor for the development of DON in patients with TED. Although dimensions of the bony orbit significantly differed between the examined groups, there was no difference predisposing to the development of DON in patients with TED prior to disease-related bony orbital changes.
In other studies apical crowding was graded on a categorical scale or by forming the ratio of the diameters of the horizontal or vertical extraocular muscles and the diameter of the bony orbit 20,21 . Other authors calculated the ratio between soft-and fat tissue around the apex area to form a volumetric apical crowding index 16 .
To our knowledge, the underlying study is the only one including both, measurements of individual muscle volumes as well as of the bony orbit. Furthermore, our collective of DON patients seems to be the largest hitherto analyzed. Finally, previously published research ignored the unaffected orbits of patients with DON, which in our study were analyzed as a separate subgroup, as it cannot be excluded that relevant anatomical differences prior to disease manifestation in orbits of patients with DON and those without exist. www.nature.com/scientificreports/ With the medial orbital wall being much thinner than the lateral orbital wall and the air-filled ethmoidal sinuses providing less resistance, the medial wall is more prone to bony remodeling. This thesis is supported by the fact that the change of the medial orbital angle correlates with an increase in muscle volume, especially that of the medial rectus muscle as shown in the TED + DON-group. Our findings regarding muscle volume are consistent with previously published research 13,15 , showing the volume or thickness of the medial rectus muscle to be the most significant predictor for the development of DON, which is most likely due to its close anatomic relation to the optic nerve in the optic canal 15 . At some point during the process of muscle enlargement, compensatory mechanisms of the orbit (mainly the expansion into the ethmoidal sinuses) seemed to exceed their limits leading to a critical increase of pressure within the orbit and ultimately optic nerve damage.
Subjects in the TED + DON group showed a distinctly increased medial orbital angle compared to the TED group. The angles of both groups were also significantly increased compared to the control group. This is in Table 1. Muscle and angle measurements of the different groups and ophthalmological patients' characteristics. p-values between TED + DON and TED group (t-test or Fisher exact test). All descriptives values are mean ± SD or absolute values (%).   12,13 . Adding medial orbital angle change to our multivariate analysis, however, did not improve the diagnostic accuracy. This suggests that the increased bowing of the medial wall serves as a surrogate parameter for the increase in muscle volume. Interestingly, this finding is contrary to previous studies 14,15 , in which there was no significant difference in medial orbital wall curvature between these groups. Reasons for this might be the use of subjective evaluation methods to assess the bowing of the medial orbital wall 15 , the higher number of subjects in our DON group, or the extent of muscle enlargement inherited by them. Previous findings describing an increase of the lateral wall angle in patients suffering from DON could not be replicated in our study. While subjects allocated to the TED + DON group had a lateral wall angle larger than subjects of the TED group, the difference was not significant (p = 0.6). Reasons for this could be the larger sample size of our study, the different patient ethnicity, group composition or even more increased bowing of the medial wall resulting in a reduction of pressure on the lateral wall. The fact that lateral orbital wall remodeling occurs later during disease progression could also explain this finding.
Unaffected (ua) orbits of DON patients did not differ markedly from orbits of the TED group regarding the dimension of the bony orbit. Despite the small sample size and the assumption, that both orbits developed more or less equally, this data might suggest that there are no characteristic bony differences or that these are too minuscule to measure, which would affect the development of DON patients prior to thickening of the muscles.  Table 2. Pearson correlation between the curvature of the medial orbital wall as well as the extent of proptosis and muscle volume. *Indicates p < .001. www.nature.com/scientificreports/ Besides bony orbital changes, protrusion of the eyeball is another effect of increased intra-orbital volume. Proptosis was remarkably higher in DON patients and correlated well with muscle volume ( Table 1). The scatterplot (Fig. 5) of medial rectus volume (MRV) and medial angle change (MAC) of DON and TED + DON patients demonstrates, that a sharp delineation between these two groups is not possible and that some cases (red and yellow points) develop atypically. Thus, these two groups therefore cannot be easily distinguished from the non-affected, respectively affected population on the basis of the measurements obtained and illustrate the difficulty of diagnosing DON using anatomical radiological data. Thus, alternative approaches like radiomics or diffusion tensor imaging might offer interesting approaches and are currently being investigated. However, currently, the clinical ophthalmological examination is indispensable and increases sensitivity and specificity 22 .

Distance between interzygomatic line and posterior sclera
Our data confirms the heterogeneity of the disease with several existing subtypes 23 . While most of the subjects exhibiting DON are showing a distinct increase in muscle volume, a subset shows no or barely any increase. On the other hand, our data shows a trend towards discrimination of both groups (TED and TED + DON) and in our opinion it may be advisable, that patients with known TED and a medial rectus volume higher than 0.9cm 3 should be monitored more closely disregarding the dimensions of the bony orbit, as this was the lowest value in our population associated with the development of DON (Fig. 5, lower horizontal line). The heterogeneity of TED has also been emphasized by Uddin et al., who stressed TED to be heterogeneous in its underlying pathogenesis, clinical manifestations, and response to medical and surgical treatment modalities 24 . In their manuscript they criticized that several previous categorizations of the clinical appearance of TED were dichotomous and underrepresented the heterogeneity of the disease. Consequently, Uddin et al. suggested a new classification system dividing TED into six different classes based on the phenotypic features (clinical and radiologic) and their response to different treatments.
Heterogeneity has not only been reported for clinical and radiological findings in EO, but also different imaging modalities including MRI and PET (alone or in combination with MRI or CT) have been used to study various morphological and functional parameters. In a recently published work, Weber et al. reported PET-MRI to be suited for the assessment of EO inflammation and to be a good discriminator for severe vs. mild-to-moderate EO 25 . A variety of other parameters have been analyzed in EO in numerous other studies. These were mainly morphological parameters like the volume or width of fat or muscles 26 or the optic nerve 27 , herniation of the lacrimal gland 28 , contrast agent uptake and T2-hyperintensity (as surrogate parameters for inflammation) 29,30 , quantitative MRI and T1-mapping of eye muscles 31,32 , just to name a few. Therefore imaging has a significant role in the investigation of TED. Siakallis et al., however, correctly pointed out that a consensus on the use of the different imaging modalities in the course of disease has not yet been reached 33 .
As it is uncommon to routinely perform CT or MRI of the orbit prior to the occurrence of TED, a definitive conclusion about risk factors regarding the dimensions of muscles and the bony orbit aggravating the progression from mere TED to TED + DON cannot be made based on this retrospective study. Thus, a follow up study closely www.nature.com/scientificreports/ assessing changes in muscle volume in our set of TED patients might be of interest to identify novel parameters (such as an increase in medial rectus volume in a certain amount of time) which are better suited to stratify the risk to progress from TED to TED + DON. It should be noted that the gender ratio in our study differs from other publications. In our population the proportion of males in the DON group was noticeably lower than in comparable studies 13,16,20 . It might be noteworthy that inclusion of patients with mild and inactive disease in the TED group (compared to the moderate to severe and sight-threatening cases) can be a confounding factor. As our center primarily sees patients who have a more serious course of disease and for whom a CT scan is justified, the measured values might not necessarily be comparable to those of the entire TED population. DON, in the majority of cases, is a clinical diagnosis being based on abnormalities in assessment of visual acuity, color vision/color saturation, perimetry, fundoscopy and/ or by testing of the pupillary light reaction/relative afferent pupillary defect in patients with TED. Radiological imaging while not mandatory, should aid in making a diagnosis in borderline cases or help to identify patients with an increased risk for the development of a DON.
To conclude, the underlying study shows the complexity of DON and the inherent problem of identifying patients with TED to develop DON. From our experience volumetric measurements of the medial rectus muscle seem to provide the best sensitivity and specificity for the development of DON and patients with a volume of the medial rectus muscle of more than 0.9cm 3 should be monitored closely.

Data availability
The datasets (CT images) generated during and/or analyzed during the current study are not publicly available due to data privacy reasons.